CN116655544A - Host-guest supermolecular probe for accelerating nitroreductase detection and application thereof - Google Patents

Host-guest supermolecular probe for accelerating nitroreductase detection and application thereof Download PDF

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CN116655544A
CN116655544A CN202310397094.XA CN202310397094A CN116655544A CN 116655544 A CN116655544 A CN 116655544A CN 202310397094 A CN202310397094 A CN 202310397094A CN 116655544 A CN116655544 A CN 116655544A
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nitroreductase
host
guest
probe
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焦扬
段春迎
张磊
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Dalian University of Technology
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Abstract

A host-guest supermolecule probe for accelerating nitroreductase detection and application thereof, belonging to the fields of organic metal supermolecules, biological inorganic and biological fluorescent probes. The supermolecule host-guest probe platform assembled by the metal organic cage host structure constructed based on the cofactor structure and the nitroreductase micromolecular fluorescent probe can be used for rapid quantitative detection of nitroreductase in tumors, so that the enzyme catalysis double-substrate process is simplified into a single-substrate process. So that the traditional fluorescence sensor depending on the concentration of the cofactor is not interfered by the cofactor any more, and has rapid fluorescence signal response to the detection of the target enzyme. The linear relationship between the fluorescence emission change of the host-guest probe and the nitroreductase concentration is shown, and the host-guest probe shows better sensitivity to the target enzyme than the simple small molecule probe. The host-guest supermolecule fusion strategy provides an effective tool for novel rapid quantitative detection of nitroreductase and a novel method for biological tracing and the like.

Description

Host-guest supermolecular probe for accelerating nitroreductase detection and application thereof
Technical Field
The application belongs to the field of metal organic supermolecules, bioinorganic and biological fluorescent probes, and in particular relates to a preparation method of a host-guest supermolecule probe for rapidly and quantitatively detecting nitroreductase and application of the host-guest supermolecule probe in aspects of solution, cell and living body detection.
Background
Tissue hypoxia is an important physiological index of many tumors, and is closely related to various physiological activities such as tumor growth, infection, diffusion metastasis and the like. Hypoxia in tumor tissue leads to a slightly acidic (pH 6.5-7.0) environment for cell growth, and hypoxia can lead to abnormal expression of various hypoxia enzymes associated therewith. In hypoxic tumor tissue, nitroreductase is at an over-expression level, and its content and activity are higher than normal cells. At present, a plurality of fluorescent probes aiming at nitroreductase are developed for tumor cell imaging and cancer diagnosis, and the fluorescent probes are activated by reduction of the nitroreductase which is over-expressed in hypoxic tumor cells, so that obvious fluorescent signal change is generated from normal cells, and thus early cancer diagnosis is realized. Nitroreductase enzymes consume the cofactor Nicotinamide Adenine Dinucleotide (NADH) through a "ping-pong" mechanism to reduce a variety of nitro compounds, which is the rationale for designing fluorescent probes for these nitroreductases. Despite the great progress made in the research related to fluorescent probes for assessing nitroreductase content, they generally require several tens of minutes of reaction to reach fluorescence emission equilibrium even in the presence of a large excess of NADH in solution (tens to hundreds of molar amounts). In this regard, it will significantly affect the detection efficiency and accuracy of nitroreductase. The concentration and expression level of nitroreductase typically varies widely among different cells and tumors, and even among the same cells. When a certain amount of conventional nitrofluorescent probe is used for detection, the co-factors NADH and nitroreductase with unknown concentrations can cause the probe emission to change significantly with time, the conventional probe has difficulty in eliminating interference of NADH concentration on detection results, and the enzyme content and the fluorescent response do not show stable linear relation. Therefore, the traditional probe method for detecting nitroreductase content and accurately distinguishing the normal and disease state enzyme activity changes is still a challenging subject.
Disclosure of Invention
The application aims to provide a rapid quantitative detection method of nitroreductase in anoxic tumors by using a host-guest supermolecular probe, which comprises a preparation method and biological test application, wherein a metal organic cage complex is used as an NADH (NADH) mimic and is fused with a guest molecule to react with nitroreductase, and an original strategy of converting a ping-pong mechanism double-substrate reaction into a single-substrate reaction is provided. By reducing the substrate species, the distance between substrates is shortened, and the reaction kinetics is promoted to be changed.
The technical scheme of the application is as follows:
a fluorogenic substrate for a nitroreductase enzyme, the fluorogenic substrate having the structural formula:
wherein ,a method for preparing a fluorogenic substrate for nitroreductase comprising the steps of:
(1) Preparing a compound shown as an intermediate 1 by taking acenaphthoquinone and 4-nitroo-phenylenediamine as raw materials; the mol ratio of acenaphthoquinone to 4-nitroo-phenylenediamine is 1:1-1.1;
(2) The intermediate 1, stannous chloride and 37% hydrochloric acid are used as raw materials, concentrated hydrochloric acid is used as a solvent, and the intermediate 2 is prepared by heating and refluxing, wherein the mol ratio of the intermediate 1 to the stannous chloride is 1:4-6;
(3) Taking an intermediate 2 and a nitro substituent as raw materials, taking carbonate as alkali, and reacting in DMF at normal temperature to prepare a nitroreductase fluorogenic substrate;
the nitro substituent is p-nitrobenzyl bromide, 2- (chloromethyl) -5-nitrothiophene, 2- (chloromethyl) -5-nitrofuran or 1- (2-chloroethyl) -2-nitro-1H-imidazole;
the intermediate 2: nitro substituent: the molar ratio of carbonate is 1:1.1-1.3:2-2.2;
a host-guest supramolecular probe comprising a host moiety and a guest moiety, the guest moiety being the fluorogenic substrate of claim 1. The main body part is a metal organic cage-shaped complex, and metal ions in the metal organic cage-shaped complex are zinc ions;
the ligand precursor compound DPM of the metal organic cage complex has the following structure:
the preparation method of the ligand precursor compound DPM comprises the following steps:
(1) Preparing a compound intermediate 3 by taking 4-bromobenzoic acid and 2-morpholinoethanolamine as raw materials and N, N' -dicyclohexylcarbodiimide and 4-dimethylaminopyridine as catalysts;
the 4-bromobenzoic acid: the molar ratio of the 2-morpholinoethanolamine to the N, N' -dicyclohexylcarbodiimide to the 4-dimethylaminopyridine is 1.1-1.3:1:1.5-1.7:1-1.2;
(2) Preparing a compound intermediate 4 by taking methyl propiolate, benzaldehyde and ammonium acetate as raw materials; the ammonium acetate: methyl propiolate: the molar ratio of the benzaldehyde is 4-4.4:2-2.2:1;
(3) Preparing a compound shown as an intermediate 5 in an organic solvent by taking the intermediate 3, the intermediate 4 and alkali as raw materials;
the alkali is sodium carbonate, potassium carbonate, cesium carbonate or organic alkali;
the organic solvent is selected from acetone, N-dimethylformamide or 1, 4-dioxane;
the molar ratio of the intermediate 3 to the intermediate 4 is 1.1-1.3:1;
(4) Reacting the intermediate 5 with 80% hydrazine hydrate to obtain a compound shown as a ligand precursor compound DPM; the molar ratio of the intermediate 5 to 80% hydrazine hydrate is 1:5 to 1:10;
the metal organic cage complex is Zn 3 DPM 3 Type (2).
The preparation method of the metal organic cage complex comprises the following steps:
the self-assembly reaction is carried out by ligand precursor compounds DPM, 2-aldehyde pyridine and zinc perchlorate hydrate, wherein the mol ratio of DPM to 2-aldehyde pyridine to zinc perchlorate hydrate is 1:2:1.
The molar ratio of host moiety to guest moiety was 1:1.
The host-guest supermolecular probe is applied to quantitative detection of nitroreductase.
The host-guest supermolecular probe is applied to preparation of reagent for detecting nitroreductase
Based on the host-guest supramolecular probe structure, basic ligand building block and enzyme catalyzed reaction, as shown in fig. 1.
Wherein the host-guest probeComprising a metallo-organic cage complex host molecule portion (Zn-MPPB) and a nitroreductase fluorogenic substrate guest molecule portion (NAQA). The main body consists of ligand precursor (DPM), 2-aldehyde pyridine and metal ion (M n+ ) M of composition 3 N 3 Metal organic cage complexes. The guest moiety is linked by a fluorophore and a p-nitrobenzyl group (formula I). Principle of reaction of probe: under the action of nitroreductase, the nitrobenzene part of the host-guest probe is reduced into hydroxylamine benzene, and the structure of the fluorescent substrate is obviously changed, so that the corresponding fluorescent response is started. Metal organic cage complex as NADH mimic and fused to substrate as quasi-fractionThe substructure is catalyzed by enzyme, the double-substrate reaction of ping-pong mechanism is changed into single-substrate reaction, the distance between substrates is shortened by reducing the substrate types, and the change of reaction dynamics is promoted.
The dihydropyridine moiety of the ligand precursor DPM is structurally similar to NADH, and serves as an active part of an NADH mimic for proton and electron transfer;
the preparation method of the metal organic cage complex comprises the following steps:
(1) Using 4-bromobenzoic acid and 2-morpholinoethanolamine as raw materials, and using N, N' -Dicyclohexylcarbodiimide (DCC) and 4-Dimethylaminopyridine (DMAP) to catalyze and esterify in methylene dichloride solution to prepare a compound shown as an intermediate 3; the 4-bromobenzoic acid: the molar ratio of 2-morpholinoethanolamine to DCC to DMAP is 1.1:1:1.5:1;
(2) Preparing a compound shown in an intermediate 4 by taking methyl propiolate, benzaldehyde, ammonium acetate and glacial acetic acid as raw materials; the ammonium acetate: methyl propiolate: the molar ratio of the benzaldehyde is 4:2:1;
(3) Preparing a compound shown in an intermediate 5 by taking the intermediate 3, the intermediate 4, alkali (excessive amount is more than or equal to 2 eq) and an organic solvent as raw materials; the alkali is sodium carbonate, potassium carbonate, cesium carbonate or organic alkali; the organic solvent is selected from acetone, N-dimethylformamide or 1, 4-dioxane, and the molar ratio of the intermediate 3 to the intermediate 4 is 1.1:1;
(4) Reacting an intermediate 5 with 80% hydrazine hydrate to obtain a compound shown as DPM; the molar ratio of the intermediate 5 to 80% hydrazine hydrate is 1:5 to 1:10;
(5) Performing self-assembly reaction on DPM, 2-aldehyde pyridine and zinc perchlorate hydrate to obtain a final metal organic cage complex Zn-MPPB; the mol ratio of DPM to 2-aldehyde pyridine to zinc perchlorate hydrate is 1:2:1.
In the guest structure, the fluorophore moiety of the fluorogenic substrate of the enzyme-catalyzed reaction may be, but is not limited to, acenaphthene [1,2-b ] quinoxaline group, etc.;
the preparation method of the fluorogenic substrate comprises the following steps:
(1) Preparing a compound shown as an intermediate 1 by taking acenaphthoquinone and 4-nitroo-phenylenediamine as raw materials; the mol ratio of acenaphthoquinone to 4-nitroo-phenylenediamine is 1:1;
(2) Taking the intermediate 1, stannous chloride and 37% hydrochloric acid as raw materials, taking concentrated hydrochloric acid as a solvent, and heating and refluxing to prepare a structure of a synthetic intermediate 2 serving as a nitroreductase fluorogenic substrate; the molar ratio of the intermediate 1 to stannous chloride is 1:4.
(3) The intermediate 2, p-nitrobenzyl bromide (or groups which can be activated by nitroreductase reduction such as 2- (chloromethyl) -5-nitrothiophene, 2- (chloromethyl) -5-nitrofuran, 1- (2-chloroethyl) -2-nitro-1H-imidazole and the like) are taken as raw materials, potassium carbonate or cesium carbonate is taken as alkali, and the raw materials react in DMF at normal temperature to prepare a structure of a synthetic formula I or formula II which is taken as a nitroreductase fluorescent substrate; the intermediate 2: p-nitrobenzyl bromide: the molar ratio of potassium carbonate is 1:1.1:2.
The preparation method of the host-guest supermolecular probe comprises the following steps:
1) The dihydropyridine ligand precursor DMP, 2-aldehyde pyridine and the zinc perchlorate hydrate which is coordinated to be required are respectively dissolved in acetonitrile solvent together for fully mixing and stirring, the metal organic complex is precipitated by solvent volatilization crystallization, solvent diffusion crystallization or adding small polar miscible solvent, and the structure M is confirmed by nuclear magnetism, mass spectrum or single crystal structure 3 N 3 M is a metal ion and N is a dihydropyridine ligand;
2) Mixing the metal organic complex and nitroreductase fluorogenic substrate in solution to obtain host-guest probe, and making one-dimensional nuclear magnetism 1 H- 1 H NOESY spectrum, two-dimensional 1 H- 1 H NOESY、 1 H- 1 The formation of host and object is determined by nuclear magnetic means such as H DOSY; meanwhile, means such as high-resolution mass spectrum, host-object ultraviolet titration, job's plot and the like verify the stability of the host-object probeAnd (5) forming.
The application of the host-guest probe in detecting nitroreductase in a solvent.
A host-guest supermolecular probe for rapid quantitative detection of nitroreductase in solvent comprises the following steps: fluorescence titration experiments, UV titration and fluorescence kinetics experiments were performed with the addition of a host-guest probe (0-5. Mu.M) to a freshly prepared nitroreductase HEPES buffer (10.0 mM, pH 6.8,25 ℃). The supermolecular probe formed by the main metal organic cage with the function of the cofactor NADH and the substrate molecule with the fluorescence indication function fuses the substrate and the coenzyme simulant into an excimer structure, so that the double-substrate catalytic process of the original nitroreductase on the coenzyme NADH and the nitro substrate is optimized into a single-substrate process. The detection of the nitroreductase realizes rapid balance and is not influenced by the concentration of NADH any more, so that the detected fluorescence intensity change is only related to the nitroreductase content change, and the rapid quantitative detection of the nitroreductase is realized.
The application of the host-guest probe in detecting nitroreductase in cells.
A host-guest supermolecular probe for the rapid quantitative detection of nitroreductase in cells comprises the following steps: DMEM medium (FBS 10% added, double antibody 1%) was used to culture MCF-7 and 231 cells, and 1640 medium (FBS 10% added, double antibody 1%) was used to culture a549 cells. And (3) culturing cells for 6-12h under normal oxygen conditions, anaero Pack and Anaero Pouch-Micro Aero (Mitsubishi Gas Chemical Company, inc.), and then incubating for 5min by using a host-guest supermolecule probe to perform laser confocal fluorescence imaging experiments of the cells under different oxygen contents. The same oxygen content environment (e.g. both at 0.1% O 2 ) The cells are cultured for 6-12h, then the host-guest supermolecule probe is used for carrying out different incubation time (such as 0-20 min) on the cells, and the time-varying experiment of the fluorescence imaging of the cells is carried out.
The application of the host-guest probe in detecting nitroreductase in living animals.
A host-guest supermolecule probe for the rapid quantitative detection of nitroreductase in a mouse living body comprises the following steps: nude mice were modeled for tumor by using the desired cancer cells (A549, MCF-7), and the host-guest supramolecular probes (generally injected at concentrations and ranges of 10-200. Mu.M, 50-200. Mu.L) described in the injection of mice during the tumor stage were used to perform imaging experiments of mouse tumor fluorescence over time.
The application has the beneficial effects that:
the present application herein proposes a supramolecular probe based on the introduction of the most common cofactor NADH mimic in hypoxic enzymes into the skeleton of the metallo-organic cage Zn-MPPBThe method is used for biological tracer imaging of the catalytic activity of nitroreductase in aqueous solution and biological systems. />The excimer structure can be directly reacted with nitroreductase together, and the natural nitroreductase and the nitro substrate and NADH double-substrate enzyme catalysis process is optimized into a simpler single-substrate catalysis process. The method eliminates interference of different concentrations on detection results in anoxic enzyme detection, and the linear relationship between fluorescence intensity and enzyme content is expected to reach rapid balance. Meanwhile, the substrate is contained in the main cage containing NADH, so that the transfer time of external NADH can be reduced, the distance between the substrate and the cofactor active site can be shortened, and the rapid signal response to the content level of the hypoxic enzyme can be realized.
The host-guest supermolecule strategy can enhance the response speed of the probe and has stronger detection capability. Through rational design, a multifunctional metal-organic cage with a certain size of hydrophobic cavity and unique chemical conversion capability, together with a guest probe molecule, acts with nitroreductase, will eliminate the excessive demand for NADH, promote delivery of the probe, and accelerate the reaction to the enzyme.
The NADH mimic is integrated into the framework of the metallo-organic cage, rendering the metallo-organic cage NADH capable. The specific nitroreductase fluorescent probe is screened as a guest molecule, the NADH simulator metal organic cage is taken as a host molecule, and the host molecule and the NADH simulator metal organic cage are assembled to form the host-guest probe, so that the electron transfer process between the host cage and the guest molecule can be effectively promoted. Hydrophobic guest molecules are stably encapsulated in the hydrophobic cavity of the metal organic cage through hydrophilic and hydrophobic actions and intermolecular interaction forces, so that an excimer structure is formed together. The excimer structure as a whole, through the single substrate process with nitroreductase reaction, has improved the reaction rate. Whereas the traditional small molecule probe and NADH react with nitroreductase through a double-substrate reaction process of a ping-pong mechanism, more time is required. By introducing amide and morpholine groups to finely adjust the cage skeleton, the hydrogen binding site is increased, the water solubility and the biocompatibility are improved, and therefore the interaction with the probe and the nitroreductase is better.
To some extent, the metal-organic cage can be used as a high-efficiency carrier to transmit the probe to the nitroreductase, and can also be used as a cofactor mimic of the nitroreductase to reduce the probe, so that the versatility and key connection effect of the metal-organic cage on the guest probe molecule and the nitroreductase are shown. These advantages weaken the interference of cofactor NADH to nitroreductase measurement results, accelerate detection efficiency, enable the host-guest probe to rapidly and accurately quantitatively analyze nitroreductase, and can be used for distinguishing and quantitatively detecting different enzyme activities in cells under normal and disease states.
In the application, the metal organic cage and the guest molecule are obviously optimized and adjusted compared with the prior structure. Each ligand of the metal organic cage is introduced with a p-formamide phenyl part, the tail chain of the ligand is increased, the cavity size of the metal organic cage is enlarged, and simultaneously, the introduced amide groups increase the hydrogen bonding sites. This allows better containment of guest molecules by optimizing cavity size, increasing hydrogen bonding sites. The guest substrate molecule is connected with the nitroreductase trigger group by introducing a large conjugated plane fluorophore structure which is larger, more rigid and more sensitive in fluorescence signal response. The increase times of the fluorescent signals before and after the enzyme reaction are greatly enhanced compared with the prior art, so that the detection effect and the sensitivity are better.
Drawings
FIG. 1 is a schematic representation of the basic building blocks of a host-guest probe and its rapid in vivo response to nitroreductase.
FIG. 2 is a schematic diagram of a conventional deviceOne dimension of (2) 1 H- 1 H NOESY and two dimensions 1 H- 1 H NOESY profile.
FIG. 3 is a schematic diagram of a preferred embodiment of the present applicationA kind of electronic device 1 H- 1 H DOSY spectrogram, zn-MPPB and +.>Is a high resolution mass spectrum of (c).
FIG. 4 is a UV titration diagram of Zn-MPPB and NAQA and a UV titration diagram of Zn-MPPB and enzyme molecules.
FIG. 5 is a Zn-MPPB andfluorescence stability profile.
FIG. 6 is a diagramAnd NAQA-NADH respectively react with nitroreductase.
FIG. 7 is a schematic diagram of a preferred embodiment of the present applicationAnd NAQA-NADH, respectively, with nitroreductase.
FIG. 8 is a diagram of various interferent pairsSelectivity and competition profile of the reaction with nitroreductase.
FIG. 9 is a graph of MTT assay analysis of Zn-MPPB and NAQA toxicity to A549 and MCF-7 cells.
FIG. 10 is a schematic diagram of an embodiment of the present applicationAnd the control group is respectively subjected to fluorescence imaging images of MCF-7 cells treated by different hypoxia treatments at the same incubation time and different incubation times at the same hypoxia.
FIG. 11 is a schematic illustration of a deviceAnd flow cytometric analysis of A549 and MCF-7 cells, respectively, in the control group.
FIG. 12 is an injectionAnd NAQA images of MCF-7 tumor-forming mice fluorescence over time.
Detailed Description
The application provides a host-guest supermolecular probe based on a structure, which is used for a strategy for rapid quantitative detection of nitroreductase in hypoxic tumors, and the host-guest probe shows the characteristic of rapid quantitative detection in solvent, cells and in-vivo experiments, and is further introduced by combining with an embodiment.
A dihydropyridine ligand hydrazide precursor DPM for assembling an NADH mimic metal organic cage complex, wherein a dihydropyridine active group has the function of NADH electron transfer, and the hydrazide group is easy to coordinate and assemble with metal ions and 2-aldehyde pyridine, and the ligand precursor is a compound shown in the following figure DPM:
a method for preparing a metal organic cage complex for an NADH mimic, comprising the steps of:
(1) Using 4-bromobenzoic acid and 2-morpholinoethanolamine as raw materials, and using N, N' -Dicyclohexylcarbodiimide (DCC) and 4-Dimethylaminopyridine (DMAP) to catalyze and esterify in methylene dichloride solution to prepare a compound shown as an intermediate 3; the 4-bromobenzoic acid: the molar ratio of 2-morpholinoethanolamine to DCC to DMAP is 1.1:1:1.5:1;
(2) Preparing a compound shown in an intermediate 4 by taking methyl propiolate, benzaldehyde, ammonium acetate and glacial acetic acid as raw materials; the ammonium acetate: methyl propiolate: the molar ratio of the benzaldehyde is 4:2:1;
(3) Preparing a compound shown as an intermediate 5 by taking the intermediate 3, the intermediate 4, alkali (excessive, >2 eq) and an organic solvent as raw materials; the alkali is sodium carbonate, potassium carbonate, cesium carbonate or organic alkali; the organic solvent is selected from acetone, N-dimethylformamide or 1, 4-dioxane, and the molar ratio of the intermediate 3 to the intermediate 4 is 1.1:1;
(4) Reacting an intermediate 5 with 80% hydrazine hydrate to obtain a compound shown as DPM; the molar ratio of the intermediate 5 to 80% hydrazine hydrate is 1:5 to 1:10;
(5) Performing self-assembly reaction on DPM, 2-aldehyde pyridine and zinc perchlorate hydrate to obtain a final metal organic cage complex Zn-MPPB; the mol ratio of DPM to 2-aldehyde pyridine to zinc perchlorate hydrate is 1:2:1.
: m obtained by assembling dihydropyridine ligand and metal ion 3 N 3 And M is a metal ion, and N is a dihydropyridine ligand.
The host-guest probe adopts NADH mimic metal organic cage complex as a host part and nitrofluorogenic substrate of nitroreductase as a guest part; the host-guest probe is assembled by an NADH mimic metal organic cage complex and a nitrofluorogenic substrate, and the molar ratio of the NADH mimic metal organic cage complex to the nitroreductase fluorogenic substrate is 1:1.
The nitroreductase fluorogenic substrate has a structure of a formula I or a formula II, and the preparation method of the nitroreductase fluorogenic substrate comprises the following steps:
(1) Preparing a compound shown as an intermediate 1 by taking acenaphthoquinone and 4-nitroo-phenylenediamine as raw materials; the mol ratio of acenaphthoquinone to 4-nitroo-phenylenediamine is 1:1;
(2) Taking the intermediate 1, stannous chloride and 37% hydrochloric acid as raw materials, taking concentrated hydrochloric acid as a solvent, and heating and refluxing to prepare a structure of a synthetic intermediate 5 serving as a nitroreductase fluorogenic substrate; the molar ratio of the intermediate 4 to stannous chloride is 1:4.
(3) The intermediate 2, p-nitrobenzyl bromide (or groups which can be activated by nitroreductase reduction such as 2- (chloromethyl) -5-nitrothiophene, 2- (chloromethyl) -5-nitrofuran, 1- (2-chloroethyl) -2-nitro-1H-imidazole and the like) are taken as raw materials, potassium carbonate or cesium carbonate is taken as alkali, and the raw materials react in DMF at normal temperature to prepare a structure of a synthetic formula I or a structure of a formula II, and the structure is taken as a nitroreductase fluorogenic substrate; the intermediate 2: p-nitrobenzyl bromide: the molar ratio of potassium carbonate is 1:1.1:2.
The preparation method of the host-guest probe assembled by the metal organic cage complex and the nitroreductase fluorogenic substrate comprises the following steps:
(1) Dissolving dihydropyridine ligand precursor, 2-aldehyde pyridine and required coordination metal salt in organic solvent, mixing and stirring thoroughly, precipitating metal organic complex by solvent volatilizing crystallization, solvent diffusion crystallization or adding small polarity mutual solvent, and confirming structure M by nuclear magnetism, mass spectrum or monocrystal structure 3 N 3 M is a metal ion and N is a dihydropyridine ligand;
(2) Mixing the metal organic complex and nitroreductase fluorogenic substrate in solution to obtain host-guest probe, and making one-dimensional nuclear magnetism 1 H- 1 H NOESY spectrum, two-dimensional 1 H- 1 H NOESY、 1 H- 1 The formation of host and object is determined by nuclear magnetic means such as H DOSY; meanwhile, the stable formation of the host-object probe is verified by means of high-resolution mass spectrum, host-object ultraviolet titration, job's plot and the like.
The metal organic cage complex and the fluorogenic substrate of the nitroreductase are fused into a host-guest supermolecule probe, and the host-guest supermolecule probe is applied to quantitative detection of the nitroreductase.
The host-guest probe is applied to quantitative detection of nitroreductase in a solvent, and 0-5 mu M of the host-guest probe is added into a nitroreductase HEPES buffer solution to perform ultraviolet titration experiments and fluorescence kinetics experiments.
Example 1: synthesis preparation of host-guest supramolecular probes
The said processThe synthesis and preparation method comprises the synthesis of metal organic cage complex, the synthesis of nitroreductase fluorogenic substrate and +.>Is prepared by the following steps.
Example 1 nitroreductase fluorogenic substrate synthesis:
(1) Synthesis of compound 1 from acenaphthoquinone and 4-nitroo-phenylenediamine
Acenaphthoquinone (1.82 g,10 mmol) and 4-nitroo-phenylenediamine (1.53 g,10 mmol) are stirred in 20ml glacial acetic acid under argon at room temperature. Heating to 80 ℃ and carrying out reflux reaction for 12h. TLC was used to monitor the progress of the reaction, after the reaction was completed, the mixture was cooled to room temperature, suction-filtered, water-washed, vacuum-dried, and purified by column chromatography to give Compound 1 as a pale yellow powder, yield 2.80g, yield 93.6%. 1 H NMR(500MHz,CF 3 COOD)δ8.92(s,1H),8.44–8.31(m,3H),8.19(d,J＝9.0Hz,1H),8.12(d,J＝8.0Hz,1H),8.04(d,J＝8.0Hz,1H),7.66(dd,J＝15.8,7.7Hz,2H). 13 C NMR(101MHz,CF 3 COOD)δ150.04,146.72,144.13,134.81,131.86,131.69,130.07,129.91,125.67,125.40,124.13,122.98,121.58,121.07,120.38,118.60,118.48.
(2) Preparation of compound 2 from compound 1, concentrated hydrochloric acid and stannous chloride
Compound 1 (1.50 g,5.0 mmol) and stannous chloride (3.80 mg,20 mmol) are reacted in concentrated hydrochloric acid (40 mL) under stirring at 80℃under reflux. TLC monitored the progress of the reaction and after completion of the reaction, the mixture was cooled to room temperature. The precipitate is filtered off, washed with 1M HCl and the pH is adjusted to 5-9. Vacuum drying, column chromatography purifying to obtain brown powderCompound 2 was obtained in an yield of 1.10g and 81.8%. 1 H NMR(500MHz,DMSO-d 6 )δ8.28(d,J＝6.9Hz,1H),8.18(d,J＝6.9Hz,1H),8.14(d,J＝8.2Hz,1H),8.09(d,J＝8.2Hz,1H),7.86(d,J＝8.9Hz,1H),7.84–7.78(m,2H),7.25(dd,J＝8.9,1.9Hz,1H),7.14(s,1H),6.13(s,2H). 13 C NMR(126MHz,DMSO-d 6 )δ153.20,150.50,148.19,143.20,134.36,134.32,132.18,131.89,130.00,129.51,129.29,128.78,128.65,128.20,121.35,120.96,120.25,106.88.ESI-MS m/z:[M+H]calculated for[C 18 H 12 N 3 ] + 270.1026,found270.1032.
(3) Synthesis of NAQA from Compound 2 and Parnitrobenzyl bromide
Compound 2 (0.27 g,1.0 mmol) and potassium carbonate (0.69 mg,5.0 mmol) were dissolved in DMF (40 mL) at ambient temperature. After mixing for 10min, p-nitrobenzyl bromide (0.26 g,1.2 mmol) was added in portions and the reaction stirred for a further 12h. TLC monitored the progress of the reaction and after completion of the reaction, the reaction was cooled to room temperature. Adding into 100ml saturated saline water, precipitating solid, suction filtering, washing with clear water, and vacuum drying. Purification by column chromatography gave the compound NAQA as a yellow powder in 263mg yield in 65.0% yield. 1 H NMR(400MHz,DMSO-d 6 )δ8.25–8.18(m,4H),8.17(d,J＝8.2Hz,1H),8.12(d,J＝8.2Hz,1H),7.90(d,J＝9.0Hz,1H),7.82(ddd,J＝8.2,7.1,3.5Hz,2H),7.71(d,J＝8.8Hz,2H),7.45(t,J＝6.1Hz,1H),7.36(dd,J＝9.1,2.6Hz,1H),6.87(d,J＝2.5Hz,1H),4.66(d,J＝6.0Hz,2H). 13 C NMR(126MHz,DMSO-d 6 )δ153.19,149.31,148.54,147.93,146.56,143.18,134.74,134.35,132.04,131.75,129.94,129.56,129.51,128.91,128.78,128.48,128.10,123.66,121.50,121.04,120.56,104.27,45.69.ESI-MS m/z:[M+H]calculated for[C 25 H 17 N 4 O 2 ] + 405.1346,found405.1336.
EXAMPLE 2 nitroreductase fluorogenic substrate Synthesis
(1) Acenaphthoquinone (1.82 g,10 mmol) and 4-nitroo-phenylenediamine (1.53 g,10 mmol) are stirred in 20ml glacial acetic acid under argon at room temperature. Heating to 80 ℃ and carrying out reflux reaction for 12h. TLC monitors the progress of the reaction, after the reaction is completed, the mixture is cooled to room temperature, suction filtered, water washed, dried in vacuo, and purified by column chromatography to give Compound 1.
(2) Compound 1 (1.50 g,5.0 mmol) and stannous chloride (3.80 mg,20 mmol) are reacted in concentrated hydrochloric acid (40 mL) under stirring at 80℃under reflux. TLC monitored the progress of the reaction and after completion of the reaction, the mixture was cooled to room temperature. The precipitate is filtered off, washed with 1M HCl and the pH is adjusted to 5-9. Drying in vacuo to give compound 2.
(3) Compound 2 (0.27 g,1.0 mmol) and potassium carbonate (0.69 mg,5.0 mmol) were dissolved in DMF (40 mL) at ambient temperature. After mixing for 10min, 2- (chloromethyl) -5-nitrothiophene (0.21 g,1.2 mmol) was added in portions and the reaction stirred for a further 12h. TLC monitored the progress of the reaction and after completion of the reaction, the reaction was cooled to room temperature. Adding into 100ml saturated saline water, precipitating solid, suction filtering, washing with clear water, vacuum drying, and column chromatography to obtain the target compound. ESI-MS m/z 400.0837.
EXAMPLE 3 nitroreductase fluorogenic substrate Synthesis
(1) Acenaphthoquinone (1.82 g,10 mmol) and 4-nitroo-phenylenediamine (1.53 g,10 mmol) are stirred in 20ml glacial acetic acid under argon at room temperature. Heating to 80 ℃ and carrying out reflux reaction for 12h. TLC monitors the progress of the reaction, after the reaction is completed, the mixture is cooled to room temperature, suction filtered, water washed, dried in vacuo, and purified by column chromatography to give Compound 1.
(2) Compound 1 (1.50 g,5.0 mmol) and stannous chloride (3.80 mg,20 mmol) are reacted in concentrated hydrochloric acid (40 mL) under stirring at 80℃under reflux. TLC monitored the progress of the reaction and after completion of the reaction, the mixture was cooled to room temperature. The precipitate is filtered off, washed with 1M HCl and the pH is adjusted to 5-9. Drying in vacuo to give compound 2.
(3) Compound 2 (0.27 g,1.0 mmol) and potassium carbonate (0.69 mg,5.0 mmol) were dissolved in DMF (40 mL) at ambient temperature. After mixing for 10min, 1- (2-chloroethyl) -2-nitro-1H-imidazole (0.21 g,1.2 mmol) was added in portions and the reaction stirred for a further 12H. TLC monitored the progress of the reaction and after completion of the reaction, the reaction was cooled to room temperature. Adding into 100ml saturated saline water, precipitating solid, suction filtering, washing with clear water, vacuum drying, and column chromatography to obtain the target compound. ESI-MS m/z 394.1044.
EXAMPLE 4 Metal organic cage Complex Zn-MPPB Synthesis
The synthetic route is as follows:
(1) Preparation of compound 3 from 4-bromobenzoic acid and 2-morpholinoethanolamine
4-Bromobenzoic acid (2.10 g,10 mmol), DCC (3.09 g,15 mmol) and DMAP (1.22 g,10 mmol) were stirred in 40ml THF in ice-bath. Then 2-morpholinoethanol (1.30 g,10 mmol) was added slowly in portions and the reaction was continued in an ice bath with stirring to room temperature for 16h. TLC was used to monitor the progress of the reaction, and after the completion of the reaction, the reaction solution was poured into 100ml of saturated brine. Extracting with dichloromethane, washing with saturated sodium carbonate, washing with clear water, and vacuum drying. Purification by column chromatography gave compound 3 as a white powder in a yield of 3.01g and 96%. 1 H NMR(400MHz,DMSO-d 6 )δ8.50(t,J＝5.5Hz,1H),7.78(d,J＝8.5Hz,2H),7.68(d,J＝8.5Hz,2H),3.59–3.54(m,4H),3.41–3.33(m,4H),2.46(t,J＝7.0Hz,2H),2.41(s,2H). 13 C NMR(126MHz,DMSO-d 6 )δ165.71,134.16,131.74,129.73,125.25,66.68,57.74,53.76,37.11.ESI-MS m/z:[M+H]calculated for[C 13 H 18 BrN 2 O 2 ] + 313.0546,found 313.0551.
(2) Preparation of Compound 4 from ammonium acetate, benzaldehyde and methyl propiolate
Methyl propionate (1.68 g,20 mmol), benzaldehyde (1.06 g,10 mmol) and ammonium acetate (2.31 g,30 mmol) were refluxed in glacial acetic acid (4.0 mL) at 80℃for 12 hours. After the reaction was cooled, the solid product was filtered off with suction and taken up in Et 2 O (10 mL. Times.3) to give a crude compound as a yellow powder4, which was recrystallized from ethanol in a yield of 1.28g, 46%. 1 H NMR(400MHz,DMSO)δ7.39(s,2H),7.26–7.18(m,4H),7.15–7.08(m,1H),4.75(s,1H),3.95(br,1H),3.54(s,6H). 13 C NMR(126MHz,DMSO)δ166.75,147.11,135.08,127.95,127.50,126.15,105.57,50.82,36.85.NMR(126MHz,DMSO).ESI-MS m/z:[M+H]calculated for C 15 H 16 NO 4 + 274.1074,found 274.1074。
(3) Preparation of Compound 5 (DMCPPD) starting from Compound 3 and Compound 4
Compound 4 (3.00 g,11 mmol) was stirred at room temperature under argon for 1h in anhydrous DMF (40 ml). Compound 3 (3.13 g,10 mmol), cuI (190 mg,1.0 mmol) and pyridine-2-carboxylic acid (246 mg,2 mmol) were added separately to the reaction solution and refluxed at 130℃for 36h. The TCL detects the reaction progress, the reaction is cooled to room temperature after completion, poured into 300ml of saturated saline water and filtered by suction to obtain a solid. Washing with clear water, vacuum drying, and column chromatography purification to obtain white powder compound 5, yield 3.40g, yield 67%. 1 H NMR(400MHz,DMSO-d 6 )δ8.50(t,J＝5.4Hz,1H),7.98(d,J＝8.5Hz,2H),7.77(s,2H),7.60(d,J＝8.6Hz,2H),7.32–7.25(m,4H),7.21–7.14(m,1H),4.83(s,1H),3.61(s,6H),3.59–3.56(m,4H),3.42(dd,J＝12.7,6.4Hz,2H),2.51–2.46(m,2H),2.42(s,4H). 13 C NMR(126MHz,DMSO-d 6 )δ166.14,165.02,145.29,144.16,134.99,131.73,128.90,128.19,127.79,126.63,119.71,110.17,66.19,57.33,53.29,51.35,37.02,36.60.ESI-MS m/z:[M+H]calculated for[C 28 H 33 N 3 O 6 ] + 506.2286,fond 506.2291.
(4) Preparation of ligand precursor compound DPM from Compound 5 and hydrazine hydrate
A mixed solution of 80% hydrazine hydrate (10 ml) and compound 5 (505 mg,1.0 mmol) was refluxed with stirring at 120℃for 12 hours. The TCL detects the progress of the reaction, after completion of the reaction, it is cooled to room temperature, collected and dried in vacuo. The crude product was purified by flash column chromatography to give the DPM ligand precursor as a pale yellow solid compound in a yield of 81mg and 16%. 1 H NMR(400MHz,DMSO-d 6 )δ9.22(s,2H),8.47(t,J＝5.6Hz,1H),7.95(d,J＝8.8Hz,2H),7.63(s,2H),7.60(d,J＝8.8Hz,2H),7.27–7.19(m,4H),7.12(t,J＝6.8Hz,1H),5.17(s,1H),4.27(s,4H),3.62–3.53(m,4H),3.41(dd,J＝12.9,6.5Hz,2H),2.50–2.45(m,2H),2.42(s,4H). 13 C NMR(126MHz,DMSO-d 6 )δ166.39,165.18,145.25,144.49,129.88,128.68,128.04,128.00,127.73,126.21,117.86,117.74,112.93,66.19,57.40,53.30,36.57.ESI-MS m/z:[M+H]calculated for[C 26 H 33 N 7 O 4 ] + 506.2510,fond 506.2502.
(5) Synthesis of metal organic cage Zn-MPPB by taking compound DPM, 2-aldehyde pyridine and zinc perchlorate hydrate as raw materials
Compound DPM (101 mg,0.2 mmol), 2-aldehyde pyridine (43 mg,0.4 mmol) and zinc perchlorate hexahydrate (138 mg,0.2 mmol) were reacted in 10ml anhydrous acetonitrile at 70℃for 17h under argon. After the reaction was completed, the reaction mixture was cooled to room temperature, and diethyl ether was added to the reaction mixture while stirring vigorously to precipitate a yellow precipitate. High-speed centrifugal separation is carried out to obtain yellow solid, and vacuum drying is carried out to obtain yellow powder product Zn-MPPB, the yield is 139mg, and the yield is 93%. 1 H NMR(400MHz,DMSO-d 6 )δ12.15(br,3H),9.59(br,2H),8.85–8.31(m,12H),8.27–8.11(m,6H),8.08–7.84(m,18H),7.77–7.64(m,6H),7.59–7.19(m,18H),7.14(s,3H),5.42(s,3H),4.11–3.60(m,18H),3.54–3.05(m,18H).ESI MS m/z:[H 6 Zn 3 (MPPB) 3 ] 6+ ＝374.6128,[H 5 Zn 3 (MPPB) 3 ] 5+ ＝449.3338,[H 6 Zn 3 (MPPB) 3 (ClO 4 - )] 5+ ＝469.1253,[H 4 Zn 3 (MPPB) 3 ] 4+ ＝561.4143,[H 5 Zn 3 (MPPB) 3 (ClO 4 - )] 4+ ＝586.4042,[H 6 Zn 3 (MPPB) 3 (2ClO 4 - )] 4+ ＝611.3928,[H 3 Zn 3 (MPPB) 3 ] 3+ ＝748.2159,[H 4 Zn 3 (MPPB) 3 (ClO 4 - )] 3+ ＝781.5339,[H 5 Zn 3 (MPPB) 3 (2ClO 4 - )] 3+ ＝814.8527,[H 6 Zn 3 (MPPB) 3 (3ClO 4 - )] 3+ ＝848.1718.
Example 5Is prepared from
The obtained Zn-MPPB compound and NAQA were dissolved in deuterated DMSO, respectively, and a solution having an equal concentration was prepared and mixed at a ratio of 1:1. One-dimensional test 1 H- 1 H NOESY spectrum (FIG. 2A), two-dimensional 1 H- 1 H NOESY (FIG. 2B), 1 H- 1 H DOSY (FIG. 3A) and the like. One-dimensional 1 H- 1 H NOESY and two dimensions 1 H- 1 H NOESY spectrum results show interactions between N-H protons of NAQA and multiple protons in Zn-MPPB compounds. 1 H- 1 The H DOSY spectrum results show that after host-guest formation, the metal organic cages and guest molecules have similar diffusion coefficients. Mass spectrum (FIGS. 3B and 3C) results ESI MS m/z: [ H ] 3 Zn 3 (MPPB) 3 (NAQA)] 2+ = 882.9246 demonstrates host-guest presence. All the above experiments demonstrate that the interaction of Zn-MPPB with NAQA can form host-guest structures.
Examples 6 to 9: solvent property test based on host-guest supramolecular probes
Host-guest supramolecular probesSolvent test reference literature method of (c) and set NAQA as control group.
Example 6Stability of
Host-guest supermolecule the stability of the host-guest supermolecule probe determines its ability and scope of application. H-H interactions of Zn-MPPB and NAQA mixtures (1:1) were tested in deuterated DMSO (FIG. 2) and N-H of NAQA was found to have significant interactions with other H of Zn-MPPB. 1 H- 1 The results of the H DOSY spectrum (FIG. 3A) show that after host-guest formation, the metal organic cage and guest fractionThe sub-elements have an approximate diffusion coefficient. Nuclear magnetic resonance results indicate thatAnd a host-guest structure.Stability in aqueous environments plays a decisive role in biological testing applications. Then, microtiter and UV-visible absorbance titration experiments were performed in DMSO-HEPES buffer (v/v, DMSO 90%,25 ℃). The binding free energy was calculated to be-8.5 kcal/mol by micro-calorimetric titration and the binding constant was 1.7X10 6 M -1 . The illustrated Zn-MPPB and NAQA interactions result in a reduction of system energy, which can occur spontaneously. Binding constants of 1.1X10 were calculated by UV-visible absorption titration (FIG. 4A) 6 M -1 . The free binding energy was calculated to be-9.62 kcal/mol by molecular docking experiments. The binding constants calculated by the various experiments have good matching. In addition, for Zn-MPPB and +.>Fluorescence intensity stability experiments (fig. 5), uv absorption stability experiments, and pH uv stability tests were performed under solvent conditions. As a result, zn-MPPB and +.>The ultraviolet absorption stability of (c) was almost unchanged within 30min, and the fluorescence intensity stability was slightly changed within 20min, but the overall stability was maintained. According to the following host-guest probe and enzyme reaction kinetics experiment, cell imaging test, flow cytometry test and mouse imaging test, the test time is not more than 30min, which indicates that the host-guest probe can exist stably under the test condition. The results of these experiments demonstrate ∈ ->Can exist stably, and the host-guest probe can be used for biological test. The part of innovation utilizes various testing methods and meansTogether, the presence and stability of the host-guest probes are justified, which is indicative and normative for the application of host-guest chemistry in the biological field.
EXAMPLE 7 interaction of Zn-MPPB with nitroreductase
Interaction with nitroreductase would suggest that host-guest probe strategies might be useful for nitroreductase activity detection. First, UV titration was performed in HEPES with nitroreductase and Zn-MPPB (FIG. 4B), and the binding constant was calculated to be 6.9X10 6 M -1 . And then, circular dichroism spectrum experiments of nitroreductase and Zn-MPPB prove that the secondary and tertiary structures of the nitroreductase keep the configuration before and after Zn-MPPB is added, and the enzyme activity is not influenced. Particle size analysis shows that the average particle size of nitroreductase is 6.3nm, and after Zn-MPPB is added, the average particle size is increased to 7.7nm, which indicates that Zn-MPPB has interaction with nitroreductase and can be attached to nitroreductase. Through a molecular docking experiment, the binding free energy of NAQA binding to a nitroreductase binding pocket is calculated to be-8.68 kcal/mol, and the binding free energy of Zn-MPPB binding to the nitroreductase binding pocket is calculated to be-11.12 kcal/mol. Calculations indicate that Zn-MPPB and NAQA spontaneously proceed with reduced system energy after binding to nitroreductase. In order to verify the combination of Zn-MPPB and nitroreductase, MALDI-TOF MS test is carried out before and after adding Zn-MPPB into nitroreductase, and experiments find that before adding Zn-MPPB, characteristic peak [ M-H ] of nitroreductase mass spectrum + ]= 49698, indicating a dimeric form; after adding Zn-MPPB, characteristic peak [ M-H ] of nitroreductase-Zn-MPPB mass spectrum + ]= 50154; compared with nitroreductase mass spectrum data, the nitroreductase-Zn-MPPB complex is formed so that the nitroreductase loses one molecule of NAD + (C 21 H 28 N 7 O 14 P 2 Mr= 664.4), 2 molecules FMN (C 17 H 21 N 4 NaO 9 P, mr= 478.3) and 9 molecules H 2 O. Mass spectral data confirmed that the enzyme could bind to Zn-MPPB to form a complex. The binding constants calculated in these experiments have good matching, as demonstrated inUnder the test conditions, zn-MPPB and nitroreductase can interact, so that the host-guest supramolecular probe can be used for detecting nitroreductase activity in biological environments.
Example 8Solvent fluorescence test with NAQA and nitroreductase respectively
Nitroreductase pairs were performed in HEPES buffer (10.0 mM, pH 6.8,25 ℃)Or NAQA fluorescence response test, characterized for its activity, excitation light 440nm, emission range 500 to 700nm, fluorescence titration and fluorescence kinetics (FIGS. 6A,6B,6D and 6E) test. At->In the test of the experimental group (5 mu M of host-guest probe and 0-5 mu g/ml of nitroreductase), the host-guest probe is found to rapidly respond to nitroreductase, and fluorescence enhancement response can be completed within 30 seconds. The contrast group (NAQA 5. Mu.M, NADH 15. Mu.M, nitroreductase 0-5. Mu.g/ml) takes more than fifteen minutes, the fluorescence intensity will reach equilibrium state slowly, and the final intensity is significantly smaller than that of the host and guest probe experiment group. Based on host-guest probesAnd the fluorescence intensity of the host-guest probe is increased by more than 36 times after the reaction of the small molecular probe NAQA and nitroreductase with 5 mug/ml under the condition of the change of the intensity of the emission peak at 540nm before and after the reaction of the small molecular probe NAQA and the enzyme, and the small molecular probe is about 29 times, which shows that the host-guest probe has a certain enhancement effect on the enzyme response. Based on the fluorescence emission at 540nm, the relative fluorescence intensity was plotted against the reaction time and nitroreductase amount (FIGS. 6C and 6F), and it was found that the reason for this was +.>The fluorescent signal of the reaction of the experimental group and nitroreductase is balanced rapidly, and the fluorescence intensity is hardly time-dependentThe fluorescence intensity was only linearly dependent on the enzyme content, as a result of the change. The NAQA contrast group fluorescence intensity is related to time variation and nitroreductase content, and the relationship between fluorescence intensity and nitroreductase content cannot be accurately quantified. Description->Can be used for the ultra-fast accurate quantitative detection of nitroreductase in solution. Further fluorescence kinetics experiments and calculations (FIG. 7) using the Mies equation, compared to NAQA control group reacted with nitroreductase, < >>The maximum reaction rate and the conversion times TON of the experimental group and the nitroreductase reaction are improved by at least 10 times. The host-guest probe strategy is illustrated to enable a more rapid and accurate quantitative detection of nitroreductase than conventional small molecule probe methods.
Example 9 other biomolecular interference pairsResponsive nitroreductase fluorescence assay
The life-internal environment is quite complex, taking into account thatFor subsequent cellular and biological testing, interference from other ions or molecules must be excluded in complex physiological environments, yet still have an accurate response to nitroreductase. The common interfering species glucose (10 mM), glutathione (GSH, 1 mM), ATP (1 mM), vitamin C (1 mM), various amino acids (1 mM L-Ala, L-Cys, L-Hcy, L-His, L-Phe, L-Pro, etc.), glucose oxidase (Glu) ox ，10μg·ml -1 ) Cytochrome C (Cyt C, 10. Mu.g.ml) -1 ) And bovine serum albumin (BSA, 10. Mu.g.ml) -1 ) Respectively and->(5. Mu.M) or NAQA (5. Mu.M) for interfering substance pair +.>In response to the nitroreductase fluorescence test (as in FIG. 8). These interfering species do not cause +.A.compared to nitroreductase (5. Mu.g/ml)>And a clear fluorescence change of NAQA, indicating that they are capable of high selectivity recognition detection of nitroreductase. In addition to that, implementCompetitive assays with these interfering analytes. Will->After (5 mu M) and the interfering substances are respectively and uniformly mixed, nitroreductase is added, the fluorescence response is still ultra-fast started, the response time and the intensity are not obviously changed, and the uniformity is good. Description->Among these interferents, the selectivity is excellent, and the structural stability can be maintained.
Example 10: cell experimental test based on host-guest supermolecular probe
(1) Cytotoxicity experiments of Zn-MPPB and NAQA
After seeding the cultured cells on 96-well plates and incubating the cells with 0,1,2,5, 10, 20. Mu.M Zn-MPPB or NAQA, respectively, for 24h, the compounds were tested for toxicity to MCF-7 and A549 cells by MTT assay experiments (see FIG. 9).
(2)Imaging test of cells with different degrees of hypoxia
For different hypoxia degrees (20%, 8%,0.1% O) 2 ) Treatment of cancer cells (including MCF-7 and A549 cells) for 6hAfter incubation for 5min (5. Mu.M), confocal laser imaging was performed, excitation wavelength 458nm, and fluorescence signal collection range 500-600nm. As in FIG. 10A, use +.>(5. Mu.M) as an experimental group and NAQA (5. Mu.M) as a control group were incubated with MCF-7 cells treated with different hypoxia for 5min, respectively, and then subjected to a cell imaging test. As a result of the experiment, it was found that the fluorescence intensity of all the various cells tested was gradually increased with increasing degree of hypoxia of the cells, wherein +.>This phenomenon is more evident in the experimental group, and the fluorescence intensity is significantly higher than that of the control group. Cells with a degree of hypoxia of 0.1%,>the fluorescence of the treatment group was much higher than Chang Yang and 8%O 2 Comparison group.
(3)For 0.1% O 2 Fluorescence intensity imaging test of hypoxic cells over incubation time
For 0.1% O 2 Administration of various cancer cells (including MCF-7 and A549 cells) after 6h of hypoxic treatment(5. Mu.M) after incubation for various times (0,1,3,5, 10, 20 min), confocal laser imaging was performed with excitation wavelength 458nm and fluorescence signal collection range 500-600nm. As in FIG. 10B, use +.>(5. Mu.M) as an experimental group and NAQA (5. Mu.M) as a control group, respectively with 0.1% O 2 The fluorescence imaging test of cells was performed after incubation of the hypoxia-treated MCF-7 cells for various times. Experimental results TableObviously, as the incubation time of the drug increases, the drug is added with +.>The cell fluorescence intensity of the experimental group reaches the maximum rapidly within five minutes, and the cell fluorescence intensity is not changed obviously after the subsequent increase of the incubation time. The fluorescent intensity of the cells can be balanced after incubation for 5min, and the fluorescent intensity of the cells is not obviously changed after the subsequent incubation time is increased to 20 min; in the NAQA control group, the fluorescence intensity of cells gradually and slowly increases along with the increase of the incubation time, but is obviously weaker than that of cells in the wholeExperimental groups. The cell imaging fluorescence kinetics test result shows that the host-guest probe can rapidly carry out bioluminescence tracing on the hypoxia degree of cells and the corresponding nitroreductase.
Example 11For 0.1% O 2 Flow cytometric assay of hypoxia treated cells
Well-grown MCF-7 and A549 cells were inoculated into culture flasks and cultured for 24h until the cells were near the bottom of the flask. Then the medium is replaced with a new one, 0.1% O is carried out 2 Anaerobic treatment is carried out for 6h. Separately apply(5. Mu.M) as experimental group, NAQA (5. Mu.M) as control group was incubated for 5min, and non-dosed cells as blank group. After the treatment, the cells were dissociated, washed twice with PBS, and the collected cells were dispersed in 1ml of PBS, respectively, and subjected to flow cytometry analysis (lambda) ex =48nm, fitc 533/30 nm), as shown in the results of fig. 11, NAQA or +.>The relative fluorescence intensity of the cells in the incubated cell group was significantly stronger than that in the control group. />The relative fluorescence intensity of the cultured cells is also stronger than that of the NAQA control group, which verifies that the host-guest probe can be incubated in a short time to realize the rapid imaging capability of the nitroreductase of the cancer cells. These results are consistent with the results of confocal imaging of cells, confirming +.>The ability of the host-guest probe to be applied to flow cytometry for rapid detection of NTR in hypoxic tumor cells is demonstrated.
EXAMPLE 12 in vivo mouse laboratory test of host-guest supramolecular probes
Injecting seed tumor into armpit of nude mice, respectively establishing MCF-7 tumor-bearing mice model, and injecting after growing for 1-2 weeks Cheng Liu period(100. Mu.L, 100. Mu.M) in vivo mouse tumor fluorescence was subjected to imaging test (lambda.) over time (0,2,5, 10 min) ex /λ em =450/570 nm) (see fig. 12). NAQA (100. Mu.L, 100. Mu.M) was set as a control group. The test results show that,the tumor fluorescence intensity of the mice in the experimental group can quickly generate a stronger fluorescence signal within 20min, while the tumor fluorescence of the mice in the control group can slowly increase along with the time, and is still weak within 20 min. />Compared with the traditional biological probe, the tumor imaging device has the capability of imaging tumor bodies more quickly. />

Claims (10)

1. A fluorogenic substrate for nitroreductase, characterized in that the fluorogenic substrate has the structural formula:
wherein ,
2. the method for preparing a fluorogenic substrate for nitroreductase according to claim 1, comprising the steps of:
(1) Preparing a compound shown as an intermediate 4 by taking acenaphthoquinone and 4-nitroo-phenylenediamine as raw materials; the mol ratio of acenaphthoquinone to 4-nitroo-phenylenediamine is 1:1-1.1;
(2) The intermediate 1, stannous chloride and 37% hydrochloric acid are used as raw materials, concentrated hydrochloric acid is used as a solvent, and the intermediate 5 is prepared by heating and refluxing, wherein the mol ratio of the intermediate 1 to the stannous chloride is 1:4-6;
(3) Taking an intermediate 2 and a nitro substituent as raw materials, taking carbonate as alkali, and reacting in DMF at normal temperature to prepare a nitroreductase fluorogenic substrate;
the nitro substituent is p-nitrobenzyl bromide, 2- (chloromethyl) -5-nitrothiophene, 2- (chloromethyl) -5-nitrofuran or 1- (2-chloroethyl) -2-nitro-1H-imidazole;
the intermediate 2: nitro substituent: the molar ratio of carbonate is 1:1.1-1.3:2-2.2.
3. A host-guest supramolecular probe for accelerating nitroreductase detection, characterized by: a host-guest supramolecular probe comprising a host moiety and a guest moiety, the guest moiety being the fluorogenic substrate of claim 1.
4. A host-guest supramolecular probe for accelerating nitroreductase assay as recited in claim 3, wherein: the main body part is a metal organic cage-shaped complex, and metal ions in the metal organic cage-shaped complex are zinc ions;
the ligand precursor compound DPM of the metal organic cage complex has the following structure:
5. the host-guest supramolecular probe for accelerating nitroreductase assay according to claim 4, wherein the ligand precursor compound DPM is prepared as follows:
(1) Preparing a compound intermediate 3 by taking 4-bromobenzoic acid and 2-morpholinoethanolamine as raw materials and N, N' -dicyclohexylcarbodiimide and 4-dimethylaminopyridine as catalysts;
the 4-bromobenzoic acid: the molar ratio of the 2-morpholinoethanolamine to the N, N' -dicyclohexylcarbodiimide to the 4-dimethylaminopyridine is 1.1-1.3:1:1.5-1.7:1-1.2;
(2) Preparing a compound intermediate 4 by taking methyl propiolate, benzaldehyde and ammonium acetate as raw materials; the ammonium acetate: methyl propiolate: the molar ratio of the benzaldehyde is 4-4.4:2-2.2:1;
(3) Preparing a compound shown as an intermediate 5 in an organic solvent by taking the intermediate 3, the intermediate 4 and alkali as raw materials;
the alkali is sodium carbonate, potassium carbonate, cesium carbonate or organic alkali;
the organic solvent is selected from acetone, N-dimethylformamide or 1, 4-dioxane;
the molar ratio of the intermediate 3 to the intermediate 4 is 1.1-1.3:1;
(4) Reacting the intermediate 5 with hydrazine hydrate to obtain a compound shown as a ligand precursor compound DPM; the molar ratio of the intermediate 5 to the hydrazine hydrate is 1:5 to 1:10.
6. The host-guest supramolecular probe for accelerating nitroreductase assay of claim 4, wherein: the metal organic cage complex is Zn 3 DPM 3 Type (2).
7. The host-guest supramolecular probe for accelerating nitroreductase assay of claim 6, wherein the method of preparing the metal-organic cage complex comprises:
the self-assembly reaction is carried out by ligand precursor compounds DPM, 2-aldehyde pyridine and zinc perchlorate hydrate, wherein the mol ratio of DPM to 2-aldehyde pyridine to zinc perchlorate hydrate is 1:2:1.
8. The host-guest supramolecular probe for accelerated nitroreductase assay of any one of claims 3-7, wherein the molar ratio of host moiety to guest moiety is 1:1.
9. The use of a host-guest supramolecular probe for accelerating nitroreductase assay according to claim 8, wherein: the host-guest supermolecular probe is applied to quantitative detection of nitroreductase.
10. The use of a host-guest supramolecular probe for accelerating nitroreductase assay according to claim 8, wherein: the host-guest supramolecular probe is applied to preparation of a reagent for detecting nitroreductase.
CN202310397094.XA 2023-04-13 2023-04-13 Host-guest supermolecular probe for accelerating nitroreductase detection and application thereof Pending CN116655544A (en)

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